Summary of work: Cartilage is composed of collagen II, which serves to provide this tissue with its high tensile strength and the aggrecan proteoglycan which is responsible for the reversible compressibility of cartilage. Collagen II expression changes during aging and during the pathogenesis of degenerative joint disease (osteoarthritis, OA). The regulation of collagen II gene expression is dependent on sequences located both in the promoter region and in the first intron which contains an enhancer sequence. We have continued to optimize a functional screening assay to identify proteins present in differentiated chondrocytes that can potentially activate collagen II transcription. This includes isolating clones of rat fibroblasts that contain a reporter construct containing the collagen II promoter/enhancer linked to a hygromycin resistance gene. These cells will be transfected with an expression library from differentiated chondrocytes to look for cDNAs that can activate the regulatory sequences and confer resistance. Our analysis of regulatory sequences in the collagen II promoter has focused on a 350 bp region that is required for transcriptional activity in chondrocytes. This region contains two E-boxes which can bind a specific class of transcription factors known as bHLH proteins. Gel-shift analysis has shown that both E-boxes bind chondrocyte nuclear factors. Mutation of the most proximal E-box did not eliminate promoter activity suggesting that the E-boxes may work in a redundant manner. Constructs containing mutations of the distal E-box and double mutations are now being tested. These studies will lead to a precise determination of the transcriptional regulation of collagen II gene expression and allow the development of therapies for cartilage degeneration based on the activation of matrix protein synthesis. A polymorphism has recently been described in a coding region of the human aggrecan gene. Our preliminary work established an association between a specific allele and the occurrence of bilateral hand OA in elderly white men in the BLSA study. We are extending this study by both increasing the n-number of the original case group and by including an analysis of women with OA.